Paneth Cells Protect against Acute Pancreatitis via Modulating Gut Microbiota Dysbiosis

ABSTRACT Acute pancreatitis (AP) is usually accompanied by intestinal failure, but its mechanism is still unclear. In AP patients, the functions of Paneth cells (lysozyme, HD5, Reg3γ, and Wnt3a) decreased. Compared with AP mice, injuries and inflammation of the pancreas and ileum were aggravated in AP mice treated with dithizone (Dith) (Dith+AP mice). Intestinal permeability and bacterial translocation were also increased. 16S rRNA sequencing showed that the gut microbiota of Dith mice and Dith+AP mice exhibited a marked increase in the pathogenic bacterium Helicobacter and a significant decrease in the probiotic bacterium Blautia. Lysozyme gavage in Dith+AP mice effectively alleviated injuries of the pancreas and small intestine. The beneficial effect of lysozyme was associated with a significant increase in the probiotic bacterium Blautia and a virtual absence of the pathogenic bacterium Helicobacter. The severity of AP in antibiotic-treated mice (ABX mice) was significantly aggravated when receiving feces from Dith mice and was markedly alleviated when receiving feces from lysozyme-gavaged mice. In vitro, lysozyme increased the proliferation of enteroids by promoting the activation of the Wnt pathway and Lgr5 expression in intestinal stem cells. IMPORTANCE We demonstrate that AP patients and experimental AP mice exhibited a dysfunction of Paneth cells. Our in vivo research showed that the severity of AP was exacerbated by the long-term dysfunction of Paneth cells, which was associated with gut microbiota disorder. Restoring part of Paneth cell functions through lysozyme supplementation alleviated the severity of AP and gut microbiota dysbiosis. This study provides novel insight into the link of pancreas-gut interactions in the pathogenesis of AP, providing a new direction for the clinical treatment of intestinal complications during AP.

secreted by Paneth cells, lysozyme that played a pivotal role in establishing gut microbiome homeostasis. Mechanistically, they identified signaling molecules such as Wnt, Lgr5, and TGFbeta, important for this reversed phenotype. Furthermore, this study utilizes the organoid in vitro technique to strengthen their research findings. Overall, their transient model system indeed provides an insight into how the Paneth cell's function could be modulated against PA. What was the source of lysozyme in this study? Would it be good to use the Lysozyme knock-out model to validate the observed phenotype in this study? Is peritoneal administration of dithizone a more potent route of Paneth cells depletion than an intravenous method of choice? Corrections-Title: "Paneth cell protect against acute pancreatitis via modulating gut microbiota dysbiosis" change into "Paneth cells" Page 2, line 41: remove % from 20 (20-30%) Page 3, line 61: change "health" into healthy Page 4, line 79: rephrase "in our study" to this study Change 16s to 16S in full texts Figures labeling has to be clearer Figure S3B: Def to Def5 wherever applicable in the text, and MMP7, Cyrptidin1 panels are missing from the legend.  Figure 10F: Densitometric analysis may help to find the difference between Con to Lyz. pathology. Overall, the study is well executed, and the findings are supported by the data. Some details remain to be addressed particularly regarding overall quantification of IF images before this work can be considered for publication:

It is difficult to see some of the panels perhaps have magnified insets for all focusing on a few cells to demonstrate representative staining. An example is the probe for bacterial endotoxin this is hardly visible.
Thanks for pointing out the deficiency. We applied universal bacterial probe EUB338 to examine bacterial translocation in pancreas and small intestine. Similar fluorescent intensity was found in our study to those in several other studies (1)(2)(3). In our prior manuscript, bacterial translocation was illustrated by representative fluorescent photographs of pancreas (100× magnification) and ileum (200× magnification) and positive cells were counted for quantification. In revised version, we have appended local magnification of the typical positive staining to the upper right corner of the original pictures in Figure 4B, 4D, 8G, 8H to provide more detailed information.

What species of heliobacter are most prevalent? Were the authors able to identify H. pylori?
Perhaps I missed this information, but it would add value to discuss the diversity of heliobacter as it could suggest a preferential shift towards one species during pancreatitis.
Thanks for the reviewer's constructive questions. In our study, 16S rRNA sequencing showed that long term reduction of Paneth cells greatly increased the relative abundance of Helicobacter and significantly reduced that of Blautia in AP. These changes were significantly reversed by lysozyme treatment. Studies have shown that Helicobacter pylori, Helicobacter hepaticus, Helicobacter bilis and Helicobacter felis are the most prevalent species associated with gastrointestinal inflammation in Helicobacter.spp (4). We carried out real time PCR for these four species to confirm the specific changes of species in Helicobacter.spp.

AP+Dith, AP+Dith+lyso and Ctrl, using either bulk RNAseq of targeted sorted Paneth cells or sicRNAseq or intestinal epithelium.
Thanks for the reviewer's valuable advice. The method of isolating Paneth cells was described in an article published in Gastroenterology in 2021. Briefly, isolated crypts were incubated with TrypLE Express supplement with DNAse I (200 U/ml) and the centrifuged pellet was resuspended and incubated with CD24-PE Ab for 15 min for flow sorting (6). We prepared single cell suspension following the protocol and carried out flow cytometry. Unfortunately, we failed to isolate Paneth cells (data not shown) and bulk RNAseq for Paneth cells was not performed in our study. Due to the limitations on research funds, sicRNAseq of intestinal epithelium is beyond our current capability. Now, we are improving the method of isolation to better explore Paneth cells in AP and other gastrointestinal diseases.

Please provide cellular quantification in addition to showing representative IF images. For example, in F3 how many cells are Claudin+/DAPI+ or Occludin+/DAPI+ across conditions.
This should be performed across all IF images in all figures, for all conditions. It will help strengthen the conclusions.
Thanks for the reviewer's valuable suggestion. Your rigorous academic attitudes are worth learning. We have supplemented the quantitative analysis of all blot ( Figure S6A-B). All of original images have also been uploaded as part of the supplementary material ( Figure S7).

The biggest caveat of this study is the lack of mechanistic insight into how lysozyme is
countering the effects of AP-induced ileal microbiome population restructuring. There is a complex interplay between the host ileal epithelium and microbiome that is not explained.
However the reviewer understands that this is a work in progress, but outlining this caveat is important in the discussion section.
Thanks for the reviewer's constructive suggestion. As you mentioned, there is a complicated interplay between the intestinal epithelium and microbiota. In our study, we focused on Paneth cell and its lysozyme. Lysozyme is a cornerstone of innate immunity, killing bacteria through the hydrolysis of peptidoglycan (PG) and its high cation (7,8). Previous in vitro studies showed that lysozyme is a non-specific antimicrobial peptide, while different bacteria possessed distinct sensitivity to lysozyme (9)(10)(11). For instance, Lactobacilli was resistant to lysozyme, while Dorea was sensitive (12). In our study, changes of lysozyme are opposite to those of Helicobacter.spp and consistent with those of Blautia.spp. The relationship between lysozyme and two key genera needed to be verified by further in vitro experiments.
In addition to antimicrobial effect, lysozyme modulated innate immune responses. The sensing of lysozyme-mediated production of pathogen-associated molecular patterns including PG and lipopolysaccharide by pattern recognition receptor stimulated downstream proinflammatory signaling and the production of proinflammatory cytokines (13,14).
Lysozyme could also limit intestinal inflammation. Zhang et al showed that intestinal inflammation is associated with the failure of secretion of Paneth cell lysozyme in mouse model of Crohn's disease (15,16). Furthermore, lysozyme supplement could ameliorate intestinal inflammation of porcine colitis (17). Mechanisms of lysozyme limiting intestinal inflammation was still unclear with speculation of limited bacterial invasion and activated protective intestinal immune response.
In revised manuscript, we outlined this caveat in line 268-269, 279-286 in Discussion section.

Were there any differences across sex?
Thanks for the reviewer's question. In our study, we divided AP patients into two groups based on the course of disease to explore whether the changes of Paneth cells were associated with the course of AP. The Chi-square test on demographic data showed that there was no statistical difference in sex distribution among healthy controls, AP patients in early stage (< 72 h) and AP patients with onset time < 1 week (p = 0.31) (in Supplementary Table1). Our research ultimately concluded that AP patients presented dysfunction of Paneth cells regardless of the course of the disease (the detailed results were shown in line 60-72).
We have also regrouped AP patients and healthy controls according to gender to analyze data as you suggested. The figures below illustrate that no significant difference was found in mRNA expression of antimicrobial peptides and stem cell supporting factors across sex in AP patients (data not included in manuscript).

Reviewer #2 (Comments for the Author):
The authors recently showed that ablation of Paneth cells exacerbates pancreatic and intestinal injuries and modulates intestinal microbiota in rats with acute pancreatitis (Guo Y et al, Mediators Inflammation 2019). In the current study, they investigated the role of gut microbiota -Paneth cells interactions in a mouse acute pancreatitis (AP) model. Firstly they found that patients with AP had decreased Paneth cells and lower expression of AMP (antimicrobial peptides) genes including lysosome, which was consistent with three mouse models of AP. They established a long-term (i.e., 15 days) reduction of Paneth cells in the L-arginine AP model, which showed increased pancreatic and ileal injuries, intestinal permeability, gut dysbiosis, and bacterial translocation. Moreover, they found that supplementation with lysozyme ameliorated those phenotypes induced by acute pancreatitis in mice and confirmed it in an enteroid model, suggesting that therapeutic interventions targeting Paneth cells provide new strategies for treatment of intestinal complications in AP. There have been enough experiments conducted to provide the conclusion, but there are some critical information missing in the manuscript. The following points should be clarified to prove.
Major concerns/questions: (1) There are several important information missing in the manuscript. i) experimental methods for the other two AP mouse models (i.e., caerulin+LPS and Na-taurocholate), ii) experimental protocol for antibiotics-treated mice, and iii) clinical trial number. BioProject reference number was provided but it looks like sequencing files are not uploaded in

PRJNA774193.
Thanks for the reviewer's kind reminding. We were very sorry for missing several critical information. We have complemented the missing contents according to your suggestions. i) Mice in Cae+AP group were injected intraperitoneally with 100 μg/kg caerulein (MedChemExpress, CN) ten times with an hour interval between consecutive injections. After final injection, 5 mg/kg lipopolysaccharide (Sigma-Aldrich, USA) was intraperitoneally injected. In N+AP group, 2 % sodium taurocholate (Sigma-Aldrich, USA) solution at a volume of 50 μl/20 g bodyweight was infused into the biliopancreatic duct at the speed of 5 μl/min to induce AP. These experimental methods for the two AP mouse models have been added in line 314-319 in revised manuscript,; ii) Feces was collected from Con mice, Dith mice and Dith+Lyz mice. The processing of FMT suspension was done within 2 hours. 100 mg feces was resuspended in 1 mL saline and centrifuged for 5 min. The supernatant was used as FMT suspension. The mice receiving antibiotics were treated with vancomycin (0.5 mg/mL), neomycin (1 mg/mL), ampicillin (1 mg/mL), and metronidazole (1 mg/mL) (Sangon Biotech, CN) in their drinking water for 4 weeks. Mice were divided into four groups: Con group, ABX+Con group, ABX+Dith group and ABX+Dith+Lyz group. Con group received no treatment. ABX+Con group were gavaged with feces from Con mice for 1 weeks, ABX+Dith group were gavaged with 200μL FMT suspension from Dith mice for 1 weeks, and ABX+Dith+Lyz group were gavaged with 200μL FMT suspension from Dith+Lyz mice for 1 weeks.
AP was induced in mice from ABX+Con group, ABX+Dith group and ABX+Dith+Lyz group. In iiii) Thanks for careful review. The data of 16S rRNA sequencing couldn't be downloaded because of some operational errors. National Center for Biotechnology Information (NCBI) staff had assisted us in releasing the data. We have confirmed that the data could be downloaded. If necessary, the reviewer could check the data in BioProject PRJNA774193.
(2) QIIME was used to analyze 16s rRNA sequencing data, but it has not been updated anymore and replaced with QIIME2. Please reanalyze the data with QIIME2. And provide statistical methods used in the microbiome analysis.   Reviewer #3 (Comments for the Author): "Paneth cell protect against acute pancreatitis via modulating gut microbiota dysbiosis" by Fu et al, claims to describe a mechanism through which the Paneth cells regulate gut microbiota during acute pancreatitis (AP). Using in vivo model, they made an original observation that the reduction in Paneth cells leads to a high risk of AP, and 16S rRNA sequencing revealed an altered gut microbiota landscape with increased abundance of pathogenic bacteria such as Helicobacter with decreased number of beneficial bacteria, Blautia. An imbalance of gut microbiota was found due to poor support for increased intestinal permeability and bacterial translocation, and this phenotype was reversed by supplementing one of the potent antimicrobial peptides secreted by Paneth cells, lysozyme that played a pivotal role in establishing gut microbiome homeostasis. Mechanistically, they identified signaling molecules such as Wnt, Lgr5, and TGFbeta, important for this reversed phenotype. Furthermore, this study utilizes the organoid in vitro technique to strengthen their research findings. Overall, their transient model system indeed provides an insight into how the Paneth cell's function could be modulated against PA.
What was the source of lysozyme in this study? Would it be good to use the Lysozyme knock-out model to validate the observed phenotype in this study?
Thanks for the reviewer's question. In our research, we used lysozyme from chicken egg white (L6876, Sigma, USA) (line 305) referred to the study published in Gastroenterology.
Lyz1 -/mice had been used to demonstrate lysozyme could generate ligands to modulate Nod2 activation and regulated the intestinal inflammatory response (12,26). The application of knockout mice might further verify the critical role of lysozyme in AP. However, due to the limitations of budget and objective conditions, Lyz1 -/mice were not used in our study.
Supplementation of α-defensin5 effectively altered gut dysbiosis induced by Paneth cell dysfuncton in alcoholic hepatitis (21). Lysozyme supplementation prevented Escherichia coli expansion caused by Paneth cell defect during maternal isolation (25). Lysozyme could also ameliorate intestinal inflammation of colitis (17). Therefore, we speculated that supplementation of lysozyme could attenuate gut dysbiosis induced by long term reduction of Paneth cells and alleviate intestinal inflammation of AP. Our study ultimately showed lysozyme significantly reduced the severity of AP.

Is peritoneal administration of dithizone a more potent route of Paneth cells depletion than an intravenous method of choice?
Thanks for the reviewer's question. The mechanism that dithizone selectively depleted Paneth cells was that it could bind with zinc ions to form zinc chelate which leads to cell death (19).
Intraperitoneal and intravenous injection of dithizone are both widely recognized and deplete Paneth cells effectively. In the latest ten articles depletion of Paneth cells by dithizone in mice with doses ranged from 33 mg/kg to 100 mg/kg, nine studies chose intraperitoneal injection (20, 21, 24, 27-33).
Moreover, our previous study showed rats injected with dithizone (100 mg/kg body weight) via the tail vein were associated with high mortality rate (34). Therefore, we ultimately chose intraperitoneal injection of dithizone in our study.

Corrections-
Title: "Paneth cell protect against acute pancreatitis via modulating gut microbiota dysbiosis" change into "Paneth cells"   We thank the distinguished reviewer for careful review of our manuscript. We've corrected all mistakes or insufficiencies you mentioned above. Lysozyme treatment did not change the expression of inflammatory factors (TNF-α, IL-6, or IL-1β) in enteroids. We added the comparison of inflammatory factors between Con group and Lyz group in Figure 10J. Thank you for submitting your manuscript to mSystems. We have completed our review and I am pleased to inform you that, in principle, we expect to accept it for publication in mSystems. However, acceptance will not be final until you have adequately addressed reviewer #3 comments.
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• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. We demonstrated that AP patients and experimental AP mice exhibited dysfunction of Paneth cells.

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Our in vivo research showed that the severity of AP was exacerbated by long term dysfunction of Paneth intestinal environment (8,9). Paneth cells also serve as guardians of intestinal stem cells via providing 55 essential cytokines such as Wnt3a and TGFβ (10). Its abnormality is related to the progression of a variety 56 of diseases (11)(12)(13). Our previous study proved that transient ablation of Paneth cell by dithizone (Dith) 57 aggravated pancreatic and intestinal injuries in rat AP (14). An interaction exists between gut microbiota   Reg3γ, Wnt3a, Lgr5, and TGFβ were reduced at the mRNA level ( Figure S1A-B).  showed that the intestinal microbiota of AP mice were was largely separated from that of Con mice.

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Simultaneously, different bacterial communities were revealed between Con mice and Dith mice. There 122 was a partial overlap between bacterial communities in Dith and Dith+AP mice ( Figure 5E). Compared  Spearman's correlation analysis revealed that the severity of AP was inversely associated with the levels 148 of Paneth cell AMPs, where lysozyme harbored the highest correlation coefficient ( Figure S4C). Therefore, 149 we chose supplementation of lysozyme as the functional recovery of Paneth cells to restore the intestinal 150 homeostasis. 151 We observed less severe pancreatic injuries and apoptosis as well as much lower amylase level and 152 pancreatic W/D weight ratio in Lyz+Dith+AP mice than those in Dith+AP mice (p < 0.05) ( Figure 7A-D).

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The pancreatic inflammation was attenuated in Lyz+Dith+AP mice (p < 0.05), as evidenced by the decline

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The expression of the TJPs (claudin1, occludin, ZO-1) was increased following lysozyme administration  The possible significance of specific species is discussed in the Discussion.  Figure 10F-H, S6B)).

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Paraformaldehyde or liquid nitrogen.   Table S2.  were divided into four groups: Con group, ABX+Con group, ABX+Dith group and ABX+Dith+Lyz group.     The data are presented as the means ± SD; ns, no significant difference; * p ≤ 0.05.  Table S1. Clinical and demographic characteristics of AP patients. Table S2. The sequences of the primers used in this study.  Table S1: Accept Table S2: Accept Fig. S5: Accept Fig. S2: Accept